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甾醇类新药NSC67657诱导THP-1细胞分化及ICAT蛋白在细胞分化中的作用研究

Study on the mechanism of THP-1 cell differentiation induced by a new steroidal drug NSC67657
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摘要 目的 研究新的甲磺酸甾醇类药物NSC67657对白血病细胞的诱导分化作用及可能机制.方法 采用MTT法分析在不同浓度NSC67657作用下THP-1细胞的增殖水平,通过细胞表面分化抗原的检测,观察不同药物浓度、不同作用时间处理的THP-1细胞分化程度,并对完全分化细胞做形态学分析.通过RT-PCR和Western blot方法观察药物作用细胞前后β-catenin相关蛋白1(ICAT)基因和蛋白的表达情况;构建pDsRed-ICAT真核表达载体,测序后转染THP-1细胞,筛选阳性克隆,并做表达验证.采用流式细胞术,瑞特染色和超微结构观察,分析重组质粒转染细胞在药物处理前和处理24 h后THP-1细胞的分化情况.结果 通过比较可见药物处理后的THP-1细胞增殖明显受抑;细胞表面分化抗原CD14的表达水平随药物处理时间的延长和药物浓度的升高而增加,结合增殖分析,以10 μmol/L药物、连续诱导5 d为宜,细胞分化可达到90%以上.形态学观察验证了THP-1细胞在NSC67657的作用下向单核系分化.真核表达载体构建成功,电转后THP-1细胞ICAT基因和蛋白表达升高.药物作用前后,重组质粒转染THP-1细胞CD14的表达与对照组比较无明显差异;通过瑞特染色和超微结构观察,发现药物作用重组质粒转染THP-1细胞24 h后,细胞仍处初级分化阶段.结论 NSC67657可诱导THP-1细胞向单核系分化,并激活ICAT基因的表达,但仅是该基因的高表达并不 足以诱导THP-1细胞分化,也不会增加THP-1细胞对NSC67657 药物作用的敏感性. Objective To study the potential mechanism of the new steroidal drug NSC67657 induced leukemic cels differentiation.Methods Cell proliferation was assayed by MTT assay.Surface antigen CD14 on THP-1 cells treated by NSC67657 at different time different concentration,was detected by flow cytometry(FCM).The expression of beta-catenin-interacting protein 1(ICAT)gene and protein were detected by RT-PCR and Western blot.Eukaryotic expressing vector pDsRed-ICAT was constructed and transfeeted into HL60 cell line.FCM,Wright's staining and electronmicroscope were employed to analyse the difierentiation of transfected THP-1 cells after they were treated with NSC67657 for 24 hours.Results The proliferation of THP-1 cells was significantly inhibited by NSC67657 treatment.The level of CD14 expression was elerated in line with the increasing drug concentration and treatment time.10 μmol/L NSC67657 treatment for five days was the optimal condition for the induction of THP-1 cells ditierentiation,when the CD14+ THP-1cells were more than 90%.Morphological study indentified the THP-1 cells of monocvtic differentiation.The eukaryotic expressing vector pDSRed-ICAT was successfully constructed,and almost 90%positive clone could be obtained after G418 screening.Electro-transfection was employed for transfecting the vector into THP-1cells.After the transfection the expression of ICAT gene and protein was increased.On the NSC67657 treatment,there was not significant differenee in CD14 expression on transfected THP-1 cells compared to that on the control groups.After 24h treatment,the transfected THP-1 cells remained in early differentiated stage.Conclusion NSC67657 can induce THP-1 cell to monocytic differentiation and activate the expression of ICAT gene,but overexpression of ICAT itself is not sufficient to induce such differentiation.
作者 王伟佳 张秀明 温冬梅 WANG Wei-jia, ZHANG Xiu-ming , WEN Dong-mei (Zhongshan People's Hospital,Nanfang Medical University,Guangzhou 528402,China)
出处 《中华血液学杂志》 CAS CSCD 北大核心 2010年第6期366-370,共5页 Chinese Journal of Hematology
基金 重庆市重大科技专项资金(2004-27)
关键词 相关蛋白 β-catenin 甾醇类 THP-1细胞 细胞分化 单核系 Interacting protein,β-catenin Sterols THP-1 cells Cell differentiation,monocytic
作者简介 王伟佳,现在广东省中山市人民医院博士后工作站工作,528402 通信作者:张秀明,Email:snowtouching@yahoo.cn
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  • 1苏坚,贺修胜,容映晖,廖前进,苏琦,李艳兰,周健国.DADS诱导人结肠癌细胞双向电泳图谱的差异分析[J].中国药理学通报,2006,22(5):583-587. 被引量:15
  • 2Erik D H, Constance J G, Joseph W A ,et al. A sterol mesylate activator of CEBP Alpha signaling induces monocytic differentiation in human leukemia cells in vitro and in vivo [ R ]. Proc Amer Assoc Cancer Res ,2006,47, Abstract #4619 ( http ://www. aacrmeetingabstracts. org/cgi/content/abstract/2006/1 / 1085-a). 被引量:1
  • 3Shoemaker R, Glover C, Adelsberger J, et al. Induction of myeloid differentiation by a novel sterol mesylate compound ( NSC 67657 ). 18 th EORTC-NCI-AACR Symposium on "Molecular Targets and Cancer Therapeutics" [ R ]. Prague, czech republic, 2006, Abstract 492 ( http ://www. czech. cz). 被引量:1
  • 4Timchenko N A, Harris T E, Wilde M, et al. CCAAT/enhancer binding protein alpha regulates p21 protein and hepatocyte proliferation in newborn mice [ J ]. Mol Cell Biol, 1997,17 (12) :7353 - 61. 被引量:1
  • 5许文荣.临床血液学和血液检验试验指导配套教材[M].第3版,北京:人民卫生出版社,2004:1-13. 被引量:2
  • 6Hellman U, Wemstedt C, Gonez J, et al. Non-decoloured In-gel Digestion of Coomassie Blue-stained Proteins [ J ]. Anal Biochem, 1995,224( 1 ) :451 -5. 被引量:1
  • 7Kim D J, Bility M T, Billin A N, et al. PPAR bold italic beta/delta selectively induces differentiation and inhibits cell proliferation [J]. Cell Death Differ,2006,13:53 - 60. 被引量:1
  • 8Keeshan K, He B, Wouters O, et al. Tribbles Homolog 2 inactivates C/EBPα and cause acute myelogenous leukemia[ J]. Cancer Cell, 2006,10(5) :401 - 11. 被引量:1
  • 9Gerhard B, Sheo M S, Huaitian L, et al. Ras signaling enhances the activity of C/EBPα to induce granulocytic differentiation by phosphorylation of serine [ J ]. JBC,2002,29 ( 277 ) : 26293 - 9. 被引量:1
  • 10Fleck R A, Romero-Steiner S, Nahm MH. Use of HL-60 cell line to measure opsonic capacity of pneumococcal antibodies [ J ]. Clin Vacc Immunol, 2005,12 ( 1 ) : 19 - 27. 被引量:1

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